PROJECT SUMMARY Formation of a functional musculoskeletal system is dependent on the integration of contractile muscle to load-bearing tendon to bone. These tissues are initially specified independently of each other, but then seamlessly integrate as development progresses. Nevertheless, these interfaces are prone to failure from excessive mechanical loading and in many cases cannot be surgically reestablished due to differences in tissue complexity and healing. Scientists have been engineering replacement scaffolds to help restore these interfaces; however, currently used materials have poor functional outcomes. What is rarely taken into consideration in scaffold design is that tissues undergo extensive ECM remodeling during development and scar-free repair, which plays a significant role in directing cellular behavior in the formation of mature functional tissue. Researchers investigating the biology behind musculoskeletal assembly have primarily focused on the signaling pathways that regulate cellular behavior. The current paradigm is that the interfaces between muscle and tendon (myotendinous junction; MTJ) and tendon and bone (enthesis) are established through cell ? cell interactions and reciprocal signaling between the separate tissues via secreted signals. In this essay, I provide evidence that an ECM-based template, deposited before the formation of these interfaces, is also required to direct the proper combinations between muscle and tendon and bone, expanding the role of the ECM from being primarily structural to one that is also instructive. The role of the ECM during early musculoskeletal development has been largely overlooked due to a lack of tools. To address these challenges, my laboratory has been developing novel methods to map the composition and organization of the ECM over the course of musculoskeletal development. Recently, we demonstrated that the murine embryo could be labeled with non-canonical amino acids (ncAAs), which are powerful biochemical tools that enable the visualization, enrichment and identification of newly synthesized proteins within complex mixtures. In addition, my lab has pioneered optical clearing methods to image the 3D ECM architecture of biomarkers deep within developing and adult musculoskeletal tissues. Together with established knockout models in which muscle-tendon patterning is disrupted, I will use these tools to test my overall hypothesis that an ECM-based template specified before tendon and muscle differentiation is necessary for the alignment and correct integration of load-bearing muscles and tendons during vertebrate forelimb development. Understanding how the links between muscle and tendon, and tendon and bone, form will provide critical insight into the mechanisms that orchestrate musculoskeletal assembly and establish guidelines for regenerative therapies that aim to restore these interfaces in diseased and damaged tissues.